Epidermal melanocyte culture formulations

ABSTRACT

The present invention provides methods and compositions for providing graft recipients with epidermal melanocytes. Specifically, the methods and compositions of the invention provide for populations of epidermal melanocytes that may be isolated from a patient and cultured in vitro to generate a proliferating population of epidermal melanocytes that exhibit growth, migration and melanin production. The invention is based on the discovery of a culture medium composed of natural components, for culturing epidermal melanocytes that exhibit increased proliferative capacity, migratory behaviors and melanin production. The invention provides novel in vitro methods for culturing epidermal melanocytes, including those isolated from the skin of a healthy subject, to generate a proliferating population of epidermal melanocytes. The methods and compositions of the invention may be used for transplantation to treat patients having hypopigmentation or depigmentation skin disorders.

This application claims priority to the following applications: thisapplication is a continuation application of U.S. patent applicationSer. No. 10/670,971, filed Sep. 24, 2003, now U.S. Pat. No. 6,943,024.

1. INTRODUCTION

The present invention provides methods and compositions for providinggraft recipients with epidermal melanocytes. Specifically, the methodsand compositions of the invention provide for populations of epidermalmelanocytes that may be isolated from a patient and cultured in vitro togenerate a proliferating population of epidermal melanocytes thatexhibit growth, migration and melanin production. The invention is basedon the discovery of a culture medium composed of natural components, forculturing epidermal melanocytes that exhibit increased proliferativecapacity, migratory behaviors and melanin production. The inventionprovides novel in vitro methods for culturing epidermal melanocytes,including those isolated from the skin of a healthy subject, to generatea proliferating population of epidermal melanocytes. The methods andcompositions of the invention may be used for transplantation to treatpatients having hypopigmentation or depigmentation skin disorders.

2. BACKGROUND OF THE INVENTION

Skin color is determined by a combination of the pigments produced inthe skin and natural colors of the upper layers of the skin. Theepidermis, which is the outer epithelial layer of the skin, containsepidermal melanocytes which produce the skin pigmentation, referred toas melanin. In patients with hypopigmentation or depigmentation of theskin, the melanocytes in the skin are absent, destroyed ornon-functional. As a result, white patches of skin appear on differentparts of the body.

Pigmentation is not only a protective function of the melanocytes, butalso plays an important role in cutaneous aesthetics. Hypopigmentationor depigmentation of the skin results from specific disorders orconditions affecting the pigmentation system through either localdestruction or absence of melanocytes or through inhibition of theirfunction. Although this situation is clinically benign, thepsychological and social consequences can be debilitating.

Vitiligo is a specific skin pigment disorder characterized by thedestruction of epidermal melanocytes and development of patchydepigmented lesions. Current treatments, which include the use ofphotosensitizes (e.g. psoralens) with UVA radiation (PUVA) or topicalcorticosteroids, have low success rates and are generally accompanied byunpleasant side effects (Shaffali and Gawkrodger, 2000, Clin ExpDermatol 25:575).

Surgical skin grafting techniques have been applied to patients withvitiligo, including transplantation of blister tops or minigrafts fromnormal skin (Koga et al., 1988, Arch Dermatol. 124:1656; Gupta et al.,1999, Dermatol Surg 25:955; Falabella, 1984, J Dermatol Surg. Oncol.10:136; Suvanprakorn et al., 1985, J Am Acad Derm 13:968; Sarkar et al.,2001, J Dermatol 28:540; Pai et al., 2002 J. Eur Acad Dermatol Venereol16:604). These treatments have been successful where the area of thelesion is small and localized. However, in individuals who have eitherlarge depigmented patches or many depigmented patches, it is difficultto obtain enough graft to cover all the lesions (Van Geel, N. et al.,2001, Dermatology 202:162).

In order to achieve success in autologous skin transplants for largeareas of the skin, it is often necessary to obtain skin grafts withlarge number of cells, in particular melanocytes. Alternatively, one canexpand a population of autologous melanocytes in culture fortransplantation. This approach entails obtaining a population ofmelanocytes from the patient, culturing this population of cells invitro under conditions that promote cell proliferation, cell migrationand production of melanogenesis, and reintroducing the cells to thepatient's skin under conditions that promote repigmentation.

A number of different factors have been found to stimulate the growth ofmelanocytes in culture. For example, hepatocyte growth factor (HGF) is apleiotrophic growth factor family initially identified as a potentmitogen for cultured hepatocytes. HGF has been shown to stimulate growthand migration of various epithelial cells and vascular endothelial cells(Matsumoto et al., 1996, J. Biochem. 119:591; Jiang et al., 1997, HistolHistopathol 12:537). In addition, it has been shown that HGF stimulatesgrowth of cultured ocular melanocytes (Hu, 2000, Pigment Cell Res 13(Suppl. 8): 81) and in skin, HGF can be produced by the fibroblasts inthe dermis (Halaban et al., 1993, Ann New York Acad Sci 680:290).

α-Melanocyte Stimulating Factor (αMSH) is a tridecapeptide with asequence identical to the first 13 amino acids of adrenocorticotropin(ACTH). Keratinocytes appear to be a major source of the peptide (Thodyet al., 1998, Pigment Cell Res 11:265; Wakamatsu et al., 1997, PigmentCell Res 10:288). Previous reports on the effect of αMSH on culturedepidermal melanocytes have been conflicting (Halaban et al., 1988, JCell Biol 107:1611; Thody and Graham., 1998, Pigment Cell Res 11:265;Wakamatsu et al., 1997, Pigment Cell Res 10:288; Abdel-Malek et al.,1995, Proc. Natl. Acad. Sci USA 92:1789). For example, Halaban et al.,reported that αMSH failed to stimulate growth on cultured epidermalmelanocytes (Halaban et al., 1988, J Cell Bol 107:1611). However, thelack of a response in these studies may be related to the addition ofcholera toxin to the media.

Epinephrine is a potent stimulator of both α- and β-adrenergicreceptors. Epinephrine activates the β2-adrenergic receptors through thecAMP second messenger system to stimulate melanocytes. β2-adrenergicagonists stimulate growth and melanogenesis of ocular melanocytes andα-, β1- and β3-adrenergic agonists did not have any effect on growth ormelanogenesis (Hu et al., 2000, Exp. Eye Res. 71:217).

Lerner et al. (1987, J Invest Derm 89:219) described the use ofmelanocyte transplantation for the treatment of piebaldism, a geneticdisorder characterized by congenital patches of white skin and hair thatlack melanocytes. A small piece of skin was obtained from the patient byshave biopsy and the epidermal melanocytes were cultured using a culturemedium containing TPA (12-O-tetradecanoyl-phorbol-13-acetate), IBMX(3-isobutyl-1-methylxanthine), cholera toxin and newborn calf serum.Though cells were successfully transplanted to an area ofhypopigmentation, the use of TPA, a well-known tumor promoting factor,in the medium discourages its applications in human transplantations.

U.S. Pat. No. 4,757,019 also discloses the use of a culture medium forculturing human melanocytes. This medium comprises MEM, a basal medium,with 5% fetal bovine serum, phorbol 12-myristate13-acetate (PMA, alsoknown as TPA 10 ng/ml) and cholera toxin (10⁻⁸ M). Since this mediumalso contains toxic compounds, i.e., PMA and cholera toxin it would notbe appropriate for applications in human transplantation.

Olsson and Juhlin (1992, Lancet 340:981) reported the use of anepidermal melanocyte culture medium which was a formulation containingPC-1, supplemented with basic fibroblast growth factor (bFGF), dbcAMP,penicillin and streptomycin. The culture medium was used to expandisolated epidermal melanocytes for use in autologous melanocytetransplantation. This medium did not contain any serum, which isnecessary for the growth and differentiation of epidermal melanocytes.In addition, the concentration of bFGF (5 ng/ml) in the medium was toolow to stimulate adequate growth of epidermal melanocytes. Furthermore,the dbcAMP added to the medium is not a natural substance and only actsfor a short period of time. In addition, no quantitative data on thenumber of cellular divisions and melanin content of the expounded cellshave been reported, therefore, it is difficult to evaluate theefficiency of this medium. It is noteworthy, however, that Olsson andJuhlin later described the use of a non-cultured epidermal melanocytetransplantation method for the treatment of vitiligo (Olsson and Juhlin,1998, Br J Dermatol 138:644).

Additional culture media, e.g., HU16 (FIC medium) has been developed forculturing epidermal melanocytes. HU16 medium comprises F12 medium, acommercially available basal medium, supplemented with an optimalconcentration of bFGF, 3-isobutyl-1-methxanthine (IBMX), cholera toxinand fetal bovine serum. This medium has been used successfully to growepidermal melanocytes for melanocyte transplantation treatment ofvitiligo in over 120 patients in Taiwan. (Chen et al., 1999, Show ChwanMed J 1:85; Chen et al., 2000, J. Dermatol 27:434; Chen et al.; 2001, JAm Acad Dermatol 44:545)

Although the HU16 medium stimulated a great increase in melanocytegrowth, the disadvantages of this medium include that (i) the IBMX andcholera toxin contained in this medium are non-physiological,non-natural substances and cholera toxin has negative psychologicalimplications for physicians and patients, and (ii) the growth andmelanogenesis stimulating effects of this medium, although better thanfor any previously reported media, is not optimal. Thus, some patientsmay wait for prolonged periods of time for the cells to grow and a smallpercentage of patient's cells fail to ever grow well enough to meet therequirement of transplantation. Moreover, even with a successfultransplant, some patients have a lack of pigmentation at the peripheryof the transplanted region, possibly due to insufficient stimulation ofcell migration provided by this medium (Chen et al. 1999, Show Chwan MedJ 1999 1:85; Chen et al., 2000, J. Dermatol 27:434).

The requirements for medium useful for clinical cell transplantation aredifferent from those of regular culture medium. For example, medium forclinical cell transplantation should contain all natural, physiologicalsubstances, so that it will not damage the cell or pose a risk of harmto the patient after cell transplantation. Furthermore, the culturemedium should provide for cell growth, melanogenesis and migration. Noneof the previously reported culture media use for epidermal melanocytetransplantation has been found to meet these criteria, nor havequantitative tests of the effects of all these culture media on cellgrowth, melanogenesis and migration have not been reported.

Therefore, there is a need to develop novel compositions comprisingnon-toxic and/or natural and/or physiologically compatible componentsand methods of culturing epidermal melanocytes in vitro under conditionsthat promote epidermal melanocyte proliferation, as well as stimulatenormal epidermal melanocyte cell properties, including melaninproduction and cell migration.

3. SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods forculturing epidermal melanocytes. The methods and compositions of thepresent invention may be used to provide skin pigmentation to patientshaving skin disorders characterized by a lack of skin pigmentation. Suchdisorders include but are not limited to vitiligo or loss of skinpigmentation due to skin diseases such as inflammation, chemical orphysical damage. In addition, the methods of the invention may be usedin combination with dermabrasion techniques for cosmetic purposes.

The compositions of the invention relate to a culture medium comprisinga basal medium, supplemented with serum, growth factors, and agentsdesigned to elevate cAMP levels. In addition, the present inventionrelates to compositions comprising epidermal melanocytes cultured in amedium comprising a basal medium, supplemented with serum, growthfactors and agents designed to elevate cAMP levels.

The present invention further relates to methods of culturing epidermalmelanocytes for transplantation using the medium of the presentinvention to obtain a proliferating population of epidermal melanocytesthat is capable of migratory behavior and melanogenesis. The inventionis based on the observation that epidermal melanocytes cultured in themedium of the present invention possess quantitatively enhancedproliferation, migration and melanogenesis capabilities.

The present invention provides grafting methods of treating skinpigmentation disorders utilizing cultured epidermal melanocytes,comprising (1) isolating epidermal melanocytes from an individual, (2)culturing the epidermal melanocytes in the medium of the invention toobtain an expanded population of epidermal melanocytes with enhancedproliferative, migratory and melanogenesis capabilities, and (3)transplanting the expanded culture of epidermal melanocytes onto theindividual's skin.

In a specific embodiment of the invention, cultured epidermalmelanocytes may be genetically engineered, prior to transplantation, toenable them to produce a wide range of proteins, including but notlimited to, growth factors, cytokines, extracellular matrix proteins, orother biologically active molecules. In this way, any new tissue derivedfrom the transplanted epidermal melanocytes will produce the desiredbiologically active protein.

The present invention, which relates to a culture medium system withnatural and physiological components, provides a more physiologicalmicro-environment for incubation of epidermal melanocytes. In additionto serving as a source of cells for transplantation, the culture mediumsystem of the invention provides an in vitro model system for testingthe effects of various biological substances (e.g., drugs, herbs andcosmetics, etc.) on the growth, melanogenesis, migration ability andother functions of epidermal melanocytes.

4. BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A–B. Effects of bFGF (ng/ml) on cultured epidermal melanocytes.Cells were cultured with growth factor-deleted culture medium (control)or supplemented with various concentrations of bFGF (ng/ml) and culturedfor 6 days. Cell number was counted and melanin/well was measured andcompared with the controls. The results are expressed as the percentagesof the controls (3 wells in each group, Mean±SD). FIG. 1A. Cell growth.FIG. 1B. Melanin content.

FIGS. 2A–D. Effects of HGF on cultured epidermal melanocytes. Cells werecultured with growth factor-deleted culture medium (control) orsupplemented with various concentrations of HGF (ng/ml) and cultured for6 days. Cell number was counted and melanin/well was measured andcompared with the controls. The results are expressed as percentages ofthe controls (3 wells in each group, Mean±SD). FIG. 2A. Cell growth.FIG. 2B. Melanin content. FIG. 2C. Comparison of epidermal melanocytescultured with bFGF (25 ng/ml) alone or bFGF with HGF (100 ng/ml). FIG.2D. Effect of HGF on migration of epidermal melanocytes. The results areexpressed as the percentages of the controls (3 wells in each group,Mean±SD).

FIGS. 3A–B. Effects of epinephrine on cultured epidermal melanocytes.Cells were cultured with cAMP-elevating agents-deleted culture medium(control) or supplemented with various concentrations of epinephrine(ng/ml) and cultured for 6 days. Cell number was counted andmelanin/well was measured and compared with the controls. The resultsare expressed as the percentages of the controls (3 wells in each group,Mean±SD). FIG. 3A. Cell growth. FIG. 3B. Melanin content.

FIGS. 4A–B. Effects of α-MSH on cultured epidermal melanocytes. Cellswere cultured with AMP-elevating agents-deleted culture medium (control)or supplemented with various concentrations of α-MSH (ng/ml) andcultured for 6 days. Cell number was counted and melanin/well wasmeasured and compared with the controls. The results are expressed asthe percentages of controls (3 wells in each group, Mean±SD). FIG. 4A.Cell growth. FIG. 4B. Melanin content.

FIGS. 5A–C. Epidermal melanocytes cultured with various culture media(phase-contrast microscopy, ×200). FIG. 5A. Cultured with Hu74 medium.FIG. 5B. Cultured with Hu16 medium. FIG. 5C. Cultured with Olsson'smedium.

FIG. 6. Comparison of epidermal melanocytes cultured with variousculture media (Hu74 medium, Hu16 medium and Olsson's medium). Cells werecultured with various culture media for 6 days. Cell number was countedand melanin/well was measured and compared. The results are expressed asthe percentages of the H74 group (3 wells in each group Mean±SD).

FIG. 7. Comparison of various media on their long-term effects on cellgrowth of cultured epidermal melanocytes. In 3 cell lines of epidermalmelanocytes, cells were seeded into 3 flasks at the first subculture andcultured with Hu74, Hu16 and Olsson's medium, separately. Cells wereincubated and subculture until senescence. Population doubling andperiod of culture before senescence were expressed as the percentages ofthe Hu74 group (3 cell lines in each group, Mean±SD).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods forculturing epidermal melanocytes and the use of such melanocytes in skingrafts. Specifically, the methods and compositions of the invention maybe used to treat skin pigmentation disorders resulting from destructionof epidermal melanocytes or loss of epidermal melanocyte functions. Thecompositions of the invention relate to a culture medium comprisingbasal medium, supplemented with serum, growth factors, andcAMP-elevating agents. The medium of the invention differs frompreviously described medium in that it comtains only natural,physiological agents. The present invention is based on the observationthat epidermal melanocytes isolated and cultured in the medium of thepresent invention have enhanced proliferation, migration andmelanogenesis.

5.1 Isolation and Culturing of Epidermal Melanocytes

Epidermal melanocytes may be obtained from a variety of different donorsources. In a preferred embodiment, autologous epidermal melanocytes areobtained from the subject who is to receive the melanocytes. Thisapproach is especially advantageous since the immunological rejection offoreign tissue and/or a graft versus host response is avoided. In yetanother embodiment of the invention, allogenic epidermal melanocytes maybe obtained from donors who are genetically related to the recipient andshare the same transplantation antigens on the surface of theirmelanocytes. Alternatively, if a related donor is unavailable,melanocytes from antigenetically matched donors may be used.Furthermore, because melanocytes do not express HLA antigen evenallogenic melanocyte transplantation is possible.

Epidermal melanocytes may be obtained from the skin using a variety ofdifferent methods. The cells are derived from pigmented areas of thebody and preferably from the subject in need of the skin graft.Epidermal melanocytes may be obtained from a superficial skin biopsy.One or more shave biopsies of the skin are taken from normal skin areaafter local anesthesia with 0.5%–1% lidocaine. Various skin graftinstruments (e.g., a skin graft knife with a razor blade) can be used totake the biopsy (Loentz et al., 1994, J Am Acad Dermatol 30:591).

Epidermal melanocytes may also be obtained from a suction blister.Blisters may be produced by applying a vacuum of 300–400 mmHg to thesurface of the skin for 30–90 minutes. The blisters may be 100–250 mm².The tops of the blisters are then excised and processed for culturing(Chen et al., 2000, J Dermatol 27:434). Alternatively, blisters may beproduced by freezing with liquid nitrogen (Suvanprakon et al., 1985, JAm Acad Dermatol 13:968).

Epidermal sheets may be dissociated mechanically and/or treated withdigestive enzymes and/or chelating agents that weaken the connectionsbetween neighboring cells, making it possible to disperse the individualcells (Chen et al., 2000, J Dermatol 27:434). Enzymatic dissociation canbe carried out by treating the skin tissue with any of a number ofdigestive enzymes. Such enzymes include, but are not limited to,trypsin, chymotrypsin, collagenase, elastase and/or hyaluronidase.Chelating agents include, but are not limited to, EDTA. The basal layermay be teased away gently with forceps to dissociate cells from thetissue.

The cell suspension may be rinsed in medium or buffer and centrifugedone or more times to remove dissociated cells from tissue. Followingpreparation of a single cell suspension, the cells are cultured in themedium of the invention. In a preferred embodiment of the invention, thecell suspension may be grown in culture to inhibit the growth ofkeratinocytes and fibroblasts by the addition of geneticin (Halaban etal., 1984, In Vitro 20:447).

The present invention relates to a novel culture medium, referred toherein as Hu74 Medium, for use in culturing epidermal melanocytes. Themedium of the invention differs from previously described medium in thatit contains only natural, physiological agents.

The media of the invention comprises basal medium supplemented withserum, growth factors, and cAMP elevating agents. The basal medium maybe any of the standard culture medium that provides the minimalrequirements to sustain the growth of cells in culture. Such basalmedia, include but are not limited to basal amino acid/salt mixturessuch as Ham's F12, RPMI, or DMEM. Serum is added to the media inconcentrations of approximately 5–30%. Any type of animal serum may beused, including but not limited to, fetal calf, calf or human serum.Additional additives to the medium may include, for example, glucose,glutamine, vitamins and any additional additives known to those of skillin the art.

Growth factors and cytokines to be added to the basal medium includehepatocyte growth factor (HGF) and fibroblast growth factors (FGFs) suchas bFGF. The present invention is based on the observation that HGF is apotent stimulator of epidermal melanocyte migration. This effect caninduce transplanted epidermal melanocytes to migrate to the margin oftransplanted area, which is important for decreasing thehypopigmentation at the margin of the transplanted area in patients.

Furthermore, the addition of both HGF and bFGF to the medium was foundto enhance the proliferation of epidermal melanocytes indicating thatHGF has an additive growth stimulating effect in conjunction with thebFGF. Thus, addition of HGF to the medium of the invention can furtherstimulate the growth of epidermal melanocytes in vitro, therebyshortening the patient's waiting time period for the transplantation.Further, in patients wherein epidermal melanocytes do not grow well andwhere the in vitro expansion of cell number required for transplantationcannot be met, HGF can be added to improve the growth of cells therebyfacilitating transplantation.

In an embodiment of the invention, the culture medium of the inventioncontains bFGF. In a specific embodiment of the invention, theconcentration of bFGF to be added to the media is between 1 and 1000ng/ml. In a preferred embodiment of the invention, the concentration ofbFGF in the media is between 10 and 100 ng/ml. In a more preferredembodiment of the invention, the concentration of bFGF in the media isbetween 20 and 50 ng/ml.

In an embodiment of the invention, the culture medium of the inventioncontains HGF. In a specific embodiment of the invention, theconcentration of HGF to be added to the media is 10 to 1000 ng/ml. In apreferred embodiment of the invention, the concentration of HGF in themedia is 50 to 750 ng/ml. In a more preferred embodiment of theinvention, the concentration of HGF in the media is 100–500 ng/ml. In aspecific embodiment of the invention, the Hu74 Medium of the inventioncontains HGF at a concentration of 100 ng/ml.

In addition, the present invention relates to Hu74 media comprising bothHGF and bFGF. Addition of HGF to culture medium containing bFGF has beenshown to have an additive stimulating effect on epidermal melanocytes invitro.

The medium of the invention further comprises, one or more natural,non-toxic, physiological agents capable of promoting epidermalmelanocytes proliferation in culture through cellular elevation of cAMP.As described herein, the use of epinephrine and α-MSH in cAMP deletedmedium stimulated the growth, melanogenesis and migration of epidermalmelanocytes.

In an embodiment of the invention, the concentration of αMSH in themedia is between 1 and 1000 ng/ml. In a preferred embodiment of theinvention, the concentration of αMSH is between 50 and 750 ng/ml. In amore preferred embodiment of the invention, the concentration of αMSH inthe media is between 75 and 500 ng/ml. In a specific embodiment of theinvention, Hu74 Medium contains αMSH at a concentration of 100 ng/ml.

In an embodiment of the invention, the concentration of epinephrine tobe added to the media is between 1 and 3000 ng/ml. In a preferredembodiment of the invention, the concentration of epinephrine in themedia is between 500 and 2000 ng/ml. In a more preferred embodiment ofthe invention, the concentration of epinephrine in the media is between750 and 1750 ng/ml. In a specific embodiment of the invention, theconcentration of epinephrine is 1.5 μg/ml. Because epinephrine is notvery stable and is easily oxidized, antioxidants such as ascorbic acid(5 μg/ml) may be added to the Hu74 medium to stabilize the epinephrine.

In a specific non-limiting embodiment of the invention Hu74 Medium isprepared as follows. Glutamine is added to Ham's F12 (GIBCO, Carlsbad,Calif.) at a concentration of 2 mM. Gentamicin is added to the culturemedium to obtain a final concentration of 50 μg/ml. Fetal bovine serum(FBS, GIBCO, Carlsbad, Calif.) is added to the medium to obtain a 10%concentration (volume/volume). The medium containing FBS and gentamicinis stored at 4° C. and prepared fresh every two weeks. bFGF (PeproTech,Rocky Hill, N.J.) is dissolved in F12 medium to 2,500 ng/ml and storedin small vials at −70° C. The stored solution is added to the culturemedium to obtain a final concentration of 25 ng/ml once a week. HGF(PeproTech, Rocky Hill, N.J.) is dissolved in F12 medium to 10,000 ng/mland stored in small vials at −70° C. The stored solution is added to theculture medium to obtain a final concentration of 100 ng/ml once a week.An α-MSH solution is obtained by dissolving the α-MSH powder (Sigma, St.Louis, Mo.) to 100 μg/ml and is stored at −70° C. The stored solution ofα-MSH is added to the culture medium to obtain a final concentration of100 ng/ml once a week. (L)-Epinephrine bitartrate (Sigma, St. Louis,Mo.) is dissolved in PBS at 0.15 mg/ml with sodium ascorbate (Sigma, St.Louis, Mo.) (0.5 mg/ml) and stored at −70° C. Ephinedrine and ascorbicacid are added to the culture medium to obtain a final concentration of1.5 μg/ml of epinephrine and 5 μg/ml sodium ascorbate once a week.

Those of skill in the art will also recognize that one or morecommercially available substances may be used as additives orsubstitutions to the medium to support the growth of epidermalmelanocytes. Such growth may be monitored using a number of differentmethods. For example, proliferation of cells can be monitored by cellcounts using a hemocytometer or flow cytometer; cell migration can bemeasured in chemotaxis assays using, for example, a Boyden chamber; andproduction of melanin can be measured using spectrophotometry.

5.2 Administration of Epidermal Melanocytes

In a specific embodiment of the invention, the cultured epidermalmelanocytes of the invention are administered to a subject in need ofskin pigmentation. Cultured epidermal melanocytes may be grafteddirectly onto the recipient where the cells will proliferate and migrateto form new skin tissue.

Prior to transplantation of cultured epidermal melanocytes, theepidermis of the depigmented or hypopigmented area of skin is removeddown to the dermal-epidermal junction by superficial abrasion, which canbe obtained by (i) mechanical methods, e.g., abraded at 25,000revolutions per minute with a dermabrader fitted with diamond fraizes(Loentz et al., 1994, J Am Acad Dermatol 30:591–7), (ii) removal of theepidermis with a skin graft knife or laser, e.g., using a SilktouchFlashscanner attached to a Sharplan 1030 CO₂ laser at the setting of 4.5to 7 watts with a 0.2 second pulse duration (Chen et al., 2000, J.Dermatol 27:434), (iii) application of YAG laser (Pai et al., 2002, JEur Acad Dermatol Venereol 16:604), or (iv) by liquid nitrogen (Gauthieret al., 1992, J Am Acad Dermatol. 26:191).

The methods of the present invention may further be used in combinationwith human skin resurfacing techniques, including chemical peeling,dermabrasion, laser surgery, and various other methods for cosmeticpurposes or scar removal. The methods utilize chemical, mechanical andlaser techniques for removal of the upper dermal layers of the skin thatcan cause scarring and persistent redness. In an embodiment of theinvention, epidermal melanocytes are applied to the skin after a skinresurfacing treatment.

The present methods and compositions may additionally employ culturedepidermal melanocytes genetically engineered to enable them to produce awide range of functionally active biologically active proteinsincluding, but not limited to, growth factors, cytokines, hormones,inhibitors of cytokines, peptide growth and differentiation factors, andextracellular matrix proteins. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining a nucleic acid encoding the protein of interest linked toappropriate transcriptional/translational control signals. See, forexample, the techniques described in Sambrook, et al., 1992, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., andAusebel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates & Wiley Interscience, N.Y.

In addition, cultured epidermal melanocytes may be attached in vitro toa natural or synthetic matrix that provides support for the transplantedepidermal melanocytes prior to, during, and/or post-transplantation. Thematrix will have all the features commonly associated with beingbiocompatible, in that it is in a form that does not produce an adverse,or allergic reaction when administered to the recipient host. Growthfactors capable of stimulating the growth and regeneration of, forexample, skin tissue may also be incorporated into matrices. Suchmatrices may be formed from both natural or synthetic materials and maybe designed to allow for sustained release of growth factors overprolonged periods of time. Thus, appropriate matrices will both providegrowth factors and also act as an in situ scaffolding in which theepidermal melanocytes may proliferate and migrate. In preferredembodiments, it is contemplated that a biodegradable matrix that iscapable of being reabsorbed into the body will likely be most useful.

To improve epidermal melanocytes adhesion to the matrix, survival,function and/or migration of the epidermal melanocytes, the matrix mayoptionally be coated on its external surface with factors known in theart to promote cell adhesion, growth, survival or migration. Suchfactors may include cell adhesion molecules, extracellular matrixmolecules or growth factors.

Epidermal melanocytes can be administered to the recipient in aneffective amount to achieve its intended purpose. More specifically, aneffective amount means an amount sufficient to lead to the restorationof skin pigmentation or skin tissue function, thereby alleviating thesymptoms associated with disorders resulting from genetic defects ortissue damage. The progress of the transplant recipient can bedetermined using visual inspection of the transplant area to assess cellcoverage and skin pigmentation. The number of cells needed to achievethe purposes of the present invention will vary depending on the degreeof tissue damage and the size of the affected skin area. Determinationof effective amounts is well within the capability of those skilled inthe art.

Cultured melanocytes can be detached from the culture flask by variousenzymes, such as for example, by trypsin (0.05%, -EDTA (0.02%), at 37°C. for 3–10 minutes. The cell suspension is then centrifuged and thecells are resuspended with serum free Hu74 medium and placed in contactwith the denuded area of the skin surface (Chen et al., 2000, J.Dermatol 27:434–439; Olsson et al., 1992, Lancet 340:981; Olsson et al,1995, Br J Dermatol 132:587).

Alternatively, epidermal melanocytes can be administered to therecipient in one or more physiologically acceptable carriers. Carriersfor these cells may include, but are not limited to, solutions ofphosphate buffered saline (PBS), lactated Ringer's solution containing amixture of salts in physiologic concentrations or culture medium with orwithout serum. In addition, the cells may be associated with abiocompatible matrix prior to administration into the recipient host.

The methods of the present invention encompass grafting of epidermalmelanocytes onto the skin. The grafting of the epidermal melanocytesonto the desired region on the skin is accomplished by directly applyingthe cell layer to the area of the skin. In addition, growth factors orhormones may be administered to the recipient prior to and followingtransplantation for the purpose of priming the recipients tissue toaccept the transplanted cells and/or to generate an environmentsupportive of cell proliferation and/or differentiation and/or migrationand/or melanogenesis.

The site may be covered with a protective covering such as silicone(Chen et al., 2000, J. Dermatol 27:434) collagen dress (Olson et al.,1995, Br. J. Dermatol 132:587–91) or fabric gauze. The gauze may besecured with Tegaderm (3M, St. Paul, Minn.) (Chen et al., 2000, J.Dermatol 27:434; Olsson et al., 1995, Br J. Dermatol 132:587). Patientsare instructed to lie still for a period of time to allow thetransplanted cells to adhere to the transplant site.

6. EXAMPLE: CULTURE OF EPIDERMAL MAELANOCYTES

These examples further describe and demonstrate embodiments within thescope of the present invention. The examples are given solely for thepurpose of illustration and are not to be construed as limitations ofthe present invention, as many variations thereof are possible withoutdeparting from the spirit and scope of the invention.

6.1 Materials and Methods 6.1.1 Preparation of Medium

Glutamine was added to Ham's F12 (GIBCO, Carlsbad, Calif.) at aconcentration of 2 mM. Gentamicin is added to the culture medium toobtain a final concentration of 50 μg/ml. Fetal bovine serum (FBS,GIBCO, Carlsbad, Calif.) is added to the medium to obtain a 10%concentration (volume/volume). The medium containing FBS and gentamicinis stored at 4° C. and prepared fresh every two weeks. bFGF (PeproTech,Rocky Hill, N.J.) is dissolved in F12 medium to 2,500 ng/ml and storedin small vials at −70° C. The stored solution is added to the culturemedium to obtain a final concentration of 25 ng/ml once a week. HGF(PeproTech, Rocky Hill, N.J.) is dissolved in F12 medium to 10,000 ng/mland stored in small vials at −70° C. The stored solution is added to theculture medium to obtain a final concentration of 100 ng/ml once a week.(L)-Epinephrine bitartrate (Sigma, St. Louis, Mo.) is dissolved in PBSat 0.15 mg/ml with sodium ascorbate (Sigma, St. Louis, Mo.) (0.5 mg/ml)and stored at −70° C. Epinephrine and ascorbic acid is added to theculture medium to obtain a final concentration of 1.5 μg/ml ofepinephrine and 5 μg/ml sodium ascorbate.

Three deleted media were used for the study, (i) growth factor-deletedmedium, (ii) cAMP-elevating agent deleted medium and (iii)serum-depleted medium. Various testing substances were added to thethree deleted media above. These substances include bFGF, HGF,epinephrine and α-MSH. The deleted medium without the test substance wasthe negative control and the complete medium is the positive control.

Two culture media previously reported for culturing epidermalmelanocytes used in transplantation of melanocytes (without TPA) werecompared to Hu74 medium. The medium included that “Olsson's media” and“Hu16 media”.

Olsson's medium is basal medium, supplemented with bFGF (5 ng/ml),dbcAMP (0.5 mM), glutamine (2 mM), penicillin (50 u/ml) and streptomycin(0.05 mg/ml).

Hu16 medium (FIC medium) is F12 medium, a commercially available basalmedium, supplemented with bFGF (25 ng/ml), IBMX (0.1 mM), cholera toxin(10 ng/ml), glutamine (2 mM) and 10% FBS.

6.1.2. Isolation and Culture of Epidermal Melanocytes

Human skin specimens containing epidermis were obtained from a donor.The specimens were incubated in 0.25% trypsin solution (GIBCO, Carlsbad,Calif.) for 15 minutes at 37° C., followed by a 10 minute incubationwith 0.2% EDTA (Sigma, St. Louis, Mo.) solution. The epidermal sheetswere gently treated with forceps to dissociate the epidermal cells andto yield an epidermal cell suspension. The epidermal cells werecentrifuged and seeded into a 25 cm² flask with the culture medium. Theflask was incubated at 37° C. in an atmosphere of 95% humidified air and5% CO₂. Geneticin (Sigma, St. Louis, Mo.) was added (100 μg/ml) forthree days to eliminate keratinocytes and fibroblasts. The medium waschanged every three days. The melanocytes typically reached confluencein two weeks. After reaching confluence, the melanocytes were detachedusing trypsin-EDTA solution, centrifuged, diluted and seeded forsubculture. At the time of transplantation, the melanocytes weredetached using trypsin-EDTA solution, centrifuged and resuspended withF12 medium.

6.1.4 Measurement of Cultured Epidermal Melanocyte Cell Growth

Cell counting was used to evaluate the effect of the test substance ongrowth of epidermal melanocytes. Epidermal melanocytes were plated in6-well plates at a density of 1×10⁵ cells per well with medium. After 24hours, medium was replaced by the testing media. The media was changedevery three days. After six days, the cells were detached withtrypsin-EDTA solution and neutralized with culture medium with 10%serum. The cell suspension was centrifuged and the pellet wasresuspended in 1 ml F12 medium, 20 μl of the cell suspension wascollected into the tip of a Pasteur pipette and transferred to ahemocytometer. The hemocytometer was observed under an opticalmicroscope. Cells falling in four 1 mm³ areas bounded by three parallellines were counted. The cell number was obtained using Formula I shownbelow (Freshney, 1987, Culture of Animal Cells. Wiley-Liss, Inc., NewYork, 2nd edition). The average of four counts was calculated.Triplicate samples were assayed in all experiments.c=n×10⁴  FORMULA I

-   -   where c=concentration (cells/ml), n=number of cells counted.

6.1.5 Melanin Production of Cultured Epidermal Melanocytes

Melanocytes cultured as described above were detached by trypsin-EDTAsolution and counted in a hemocytometer. The cell suspensions werecentrifuged and the pellet was resuspended in 1N NaOH. Melaninconcentration was determined by measurement of optical density at 475 nmby a spectrophotometer and compared with a standard curve obtained usingsynthetic melanin (Sigma, St. Louis, Mo.). Melanin content is expressedas μg/culture or ng/cell (Hu et al., 1995, Invest. Ophthalmol Vis Sci36:931).

6.1.6 Migratory Behavior of Cultured Epidermal Melanocytes

Cell migration experiments were performed on early passages of cells in48-well Boyden chambers (Neuroprobe, Bethesda, Md.). Cultured epidermalmelanocytes were dissociated with 0.05% trypsin-EDTA (Sigma, St. Louis,Mo.) at 37° C. for 2–3 minutes, neutralized by culture medium with 10%FBS. The cells were centrifuged and resuspended in culture medium. Thelower half of the Boyden chambers contained culture medium with orwithout tested substances. An 8 μm pore cellulose nitrate filterseparated the upper and lower wells. Epidermal melanocytes (3×10⁴)suspended in 50 μl culture medium (with or without tested substances)were added to the upper wells. The chamber was incubated at 37° C. in ahumidified atmosphere at 95% air and 5% CO₂. After 6 hours, the filterwas removed from the chamber, fixed in 95% ethanol for 5 minutes, andstained with hematoxylin for 1 minute. The number of cells migratingthrough the filter and attached to the lower surface of the filter wascounted (Doerr et al., 1996, J Biol Chem 271:2443; Verdoorn et al.,1986, Arch Ophtalmol 104:1216). Triplicate samples were assayed in allexperiments.

6.1.7. Comparison of Long-Term Effects on Cell Growth by HU74 Mediumwith Other Culture Media

The long-term results of epidermal melanocytes cultured with Hu74medium, Hu16 medium and Olsson's medium were compared for threedifferent cell lines. In each cell line, the epidermal melanocytes wereseeded into three flasks at the first subculture, and cultured with 3different media. Cells were incubated and subcultured continuously,until senescence. Population doubling (PD) of each generation wascalculated from the number of cells plated and the number of cellsharvested (Hu et al., 1992, Invest Ophthalmol Vis Sci 33:2443) using thefollowing formula:PD=(log Nt−log No)/log 2  Formula II

-   -   No=cell number at the time of plating    -   Nt=cell number at the time of harvesting

The cumulative population doublings of a cell line cultured with a givenculture medium is the sum of PD in all generations (Hu et al., 1992,Invest Ophthalmol Vis Sci 33:2443). The period of epidermal melanocytescultured with each culture medium before senescence was also compared.

6.2. Results 6.2.1 Effects of bFGF on Cell Growth and Melanogenesis ofCultured Epidermal Melanocytes

Epidermal melanocytes cultured in the absence of growth factor grew veryslowly. Addition of bFGF at concentrations of 1–1,000 ng/ml caused adose dependent stimulation of cell growth (FIG. 1A). Cell number ofepidermal melanocytes cultured with bFGF was significantly greater thanthat of the controls (P<0.01 from 10–1000 ng/ml). However, bFGF did notshow any significant influence on melanin content at any concentration(P<0.05) (FIG. 1B).

6.2.2 Effects of HGF on Cell Growth, Melanogenesis and Migration ofCultured Epidermal Melanocytes

Epidermal melanocytes cultured in the absence of growth factor grew veryslow. Addition of HGF at concentrations of 1–1,000 ng/ml caused a dosedependent stimulation of cell growth (FIG. 2A). Cell number of epidermalmelanocytes cultured with HGF was significantly greater than that of thecontrols (0.05>P>0.01 at concentration of 3 ng/ml and P<0.01 at 10–1000ng/ml). HGF was also found to stimulate the production of melanin.Melanin content/well of epidermal melanocytes cultured with HGF wassignificantly greater than that of the controls (0.05>P>0.01 atconcentration of 3 ng/ml and P<0.01 at 10–1000 ng/ml) (FIG. 2B).

In epidermal melanocytes cultured with 25 ng/ml of bFGF, addition of HGF(100 ng/ml) caused a further increase of cell growth and melanincontent/well. The cell number of epidermal melanocytes cultured with HGFand bFGF was significantly greater than that cultured with bFGF alone(P<0.01). Melanin content/well in epidermal melanocytes cultured withHGF and bFGF was also significantly greater than cultured with bFGFalone. (0.05>P>0.01) (FIG. 2C).

Epidermal melanocytes cultured with HGF showed a better migratorybehavior than that of the controls. Addition of HGF at concentrations of1–1,000 ng/ml caused a dose dependent stimulation of migration (FIG.2D). Migration cell number of epidermal melanocytes cultured with HGFwas significantly greater that that of the controls (0.05>P>0.01 at aconcentration of 10 ng/ml and P<0.01 at 10–1000 ng/ml).

6.3. Effects of Epinephrine on Cell Growth and Melanogenesis of CulturedEpidermal Melanocytes

Epidermal melanocytes cultured without cAMP-elevating agents grew veryslowly and showed a relatively low melanin content/well. Addition ofepinephrine at concentrations from 1–3,000 ng/ml caused a dose dependentstimulation of cell growth (FIG. 3A). Cell number of epidermalmelanocytes cultured with epinephrine was significantly greater thanthat of the controls (0.05>P>0.01 at concentration of 3 ng/ml and P<0.01at 10–3000 ng/ml). Epinephrine also stimulated the production ofmelanin. Melanin content/well of epidermal melanocytes cultured withepinephrine was significantly greater than that of the controls(0.05>P>0.01 at concentration of 3 ng/ml and P<0.01 at 10–3000 ng/ml)(FIG. 3B).

6.2.4. Effects of α-MSH on Cell Growth and Melanogenesis of CulturedEpidermal Melanocytes

Epidermal melanocytes cultured with cAMP elevating agents grew veryslowly and showed a relatively low melanin content/well. Addition ofα-MSH at concentrations from 1–1,000 ng/ml caused a dose dependentstimulation of cell growth (FIG. 4A). Cell number of epidermalmelanocytes cultured with α-MSH was significantly greater than that ofthe controls (0.05>P>0.01 at concentration of 1 ng/ml and P<0.01 at10–1000 ng/ml). α-MSH also stimulated the production of melanin. Melanincontent/well of epidermal melanocytes cultured with α-MSH wassignificantly greater than that of the controls (0.05>P>0.01 atconcentration of 1 ng/ml and P<0.01 at 10–1000 ng/ml) (FIG. 4B).

6.2.5. Comparison of Hu74 Medium to Other Culture Media

Epidermal melanocytes cultured with Hu74 medium grew better than cellscultured in Hu16 medium and Olsson's medium. Cell number of epidermalmelanocytes cultured with Hu74 medium was significantly greater thanthat cultured in Hu16 medium and Olsson's medium (0.05>P>0.01, Hu74medium compared with Hu16 medium and P<0.01, Hu74 medium compared withOlsson's medium) (FIG. 6).

6.2.6 Comparison of Long-Term Growth Effects of Hu74 Medium with OtherCulture Media

Three cell lines of epidermal melanocytes were cultured with Hu74medium, Hu 16 medium and Olsson's medium from the first subculture andpassaged until senescence. Epidermal melanocytes cultured with Hu74medium could be passaged for 111.7±4.5 days before senescence, which wassignificantly longer than that cultured with Hu 16 medium (86.7±6.5days, 0.05>P>0.01), and was very significantly longer than that ofOlsson's medium (32.3±9.5 days, P>0.01)(FIG. 7). Epidermal melanocytescultured with Hu74 medium divided 30.4±1.91 times (cumulative populationdoubling), which was very significantly more than that cultured withHu16 medium (22.9±1.34) and Olsson's medium (4.3±0.35)(P>0.01 in Hu74medium as compared with both Hu16 or Olsson's media)(FIG. 7).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingFigures. Such modifications are intended to fall within the scope of theappended claims. Various references are cited herein, the disclosure ofwhich are incorporated by reference in their entireties.

1. A cell culture comprising epidermal melanocytes, basal medium, serum,one or more antibiotics, one or more growth factors, and one or morenatural, physiological cAMP-elevating agents, wherein the one or morenatural, physiological cAMP-elevating agents comprise epinephrine,wherein the one or more growth factors comprise hepatocyte growthfactor, and wherein the cultured epidermal melanocytes exhibitproliferative growth, melanin production and migratory behavior.